Influence of proteolysis of milk on the whey protein to total protein ratio as determined by capillary electrophoresis

Capillary electrophoresis (CE) was used to determine the whey protein to total protein ratio in raw and UHT milk samples with different degrees of proteolysis caused by storage. In raw milks, the analysis of samples taken at regular times demonstrated the influence of proteolysis in the whey protein to total protein determination, which was overestimated after 4 d of storage. In UHT milks, the overestimation of the whey protein to total protein ratio took place after 30 or 60 d of storage. However, the ratios alphaS1-CN/beta-CN and alphas1-CN/kappa-CN permitted detection of the samples of raw or UHT milk with degraded proteins. The distorted capillary electrophoretic pattern obtained for UHT milks made necessary an integration of the electropherograms in a "valley-to-valley" way. Results for raw milk samples were identical when "valley-to-valley" was compared to standard integration techniques. This CE method could be considered an alternative method to derivative spectroscopy for the determination of the whey protein to total protein of milk and could be used to detect samples with proteolysis.     

Proteolysis in ultra‐heat‐treated skim milk after exposure to multispecies biofilms under conditions modelling a milk tanker

Bacteria from milk tankers can form multispecies biofilms and produce heat‐stable enzymes. In this study, milk was exposed to multispecies biofilms in stainless steel vessels and was then used to produce ultra‐heat‐treated (UHT) milk, which was stored for 5 months. The UHT milks were assessed for microbial counts, free peptide concentration and pH. The free peptide concentration, which indicated proteolysis, was higher in UHT milk that had been exposed to multispecies biofilms than in untreated UHT milk. Biofilm formation may be promoted in milk tankers that are not properly cleaned, which may compromise the quality of the final dairy product.     

IMPROVED SHELF LIFE ESTIMATION OF UHT MILK BY PREDICTION OF PROTEOLYSIS

ABSTRACT

During growth in raw milk, many psychrotrophic bacteria produce proteases that can retain activity following ultra‐high temperature (UHT) treatment. In this study, casein and skim milk powder assays for detecting very low levels of protease in UHT milk were optimized, and the suitability of azocasein and fluorescein isothiocyanate‐casein (FITC‐casein) as substrates was investigated. The strongest correlations of protease activity with proteolysis in stored UHT milk were observed when FITC‐casein was used as substrate in the assays. Assays using casein and FITC‐casein as substrates yielded the highest activities. To determine sensitivity, crude protease was added at low concentrations to UHT milk, and the milk was assayed for progress of proteolysis over 12 months and for protease activity using the casein and FITC‐casein assays. With long assay incubation times, the FITC‐casein assay was more sensitive than the casein assay and may be suitable for detecting very low levels of protease activity and predicting progress of proteolysis in stored UHT whole milk.

PRACTICAL APPLICATIONS

This study contributes to the development and evaluation of practical assays for the detection of protease activity in the industry to identify potential premature spoilage of contaminated UHT milk before it is distributed for sale. The developed assays are also useful for assessing the quality of milk powder as active protease can persist in milk powder to cause spoilage in reconstituted milk. Although the assays require up to 14 days to complete, this is not an excessive time, compared with the time required for microbiological clearance and total shelf life of the product. High protease activity can be identified with less incubation time. The cost of protease detection assays developed during this work is quite low and, although 20 min analysis time is required per sample, the tests can be very cost efficient when run in batches, as would be expected in a commercial testing facility.

     

Effect of somatic cell counts on lipolysis,proteolysis and apparent viscosity of UHT milk during storage

In this work, lipolysis, proteolysis and viscosity of ultra‐high temperature (UHT) milk containing different somatic cell counts (SCC) were investigated. UHT milks were analysed on days 8, 30, 60, 90 and 120 of storage. Lipolysis as measured by free fatty acids increase, casein degradation and viscosity of UHT milk were not affected by SCC but increased during storage. A negative relationship was observed between SCC and casein as a percentage of true protein on the 120th day of storage, hence indicating that high SCC increases the proteolysis of UHT milk by the end of its shelf life.     

Effect of addition of CO₂ to raw milk on quality of UHT-treated milk

The objective of this study was to evaluate the effect of addition of CO(2) to raw milk on UHT milk quality during storage. Control milk (without CO(2) addition) and treated milk (with CO(2) addition up to pH 6.2) were stored in bulk tanks at 4°C for 6d. After storage, both samples were UHT processed using indirect heating (140°C for 5s). Samples were aseptically packed in low-density polyethylene pouches and stored in the dark at room temperature. Raw milk was evaluated upon receipt for physicochemical composition, proteolysis, lipolysis, standard plate count, psychrotrophic bacteria, and Pseudomonas spp. counts, and after 6d of storage for proteolysis, lipolysis, and microbial counts. After processing, UHT milk samples were evaluated for physicochemical composition, proteolysis, and lipolysis. Samples were evaluated for proteolysis and lipolysis twice a month until 120d. Peptides from pH 4.6-soluble N filtrates were performed by reversed-phase HPLC after 1 and 120d of storage. A split-plot design was used and the complete experiment was carried out in triplicate. The results were evaluated by ANOVA and Tukey's test. After 6d of storage, CO(2)-treated raw milk kept its physicochemical and microbiological quality, whereas the untreated milk showed significant quality losses. A significant increase in proteolysis occurred during 120d of storage in both treatments, but the increase occurred 1.4 times faster in untreated UHT milk than in CO(2)-treated UHT milk. In both UHT milks, the proteolysis was a consequence of the action of plasmin and microbial proteases. However, the untreated UHT milk showed higher microbial protease activity than the treated UHT milk. The addition of CO(2) to the raw milk maintained the quality during storage, resulting in UHT milk with less proteolysis and possibly longer shelf life, which is usually limited by age gelation of UHT milk.     

Detection of rennet whey solids in UHT milk by capillary electrophoresis

The fraudulent presence of rennet whey solids in UHT milk was studied by capillary electrophoresis (CE). Commercial UHT samples of different origins, genuine milk samples and milk samples adulterated with rennet whey were analysed. Linear discriminant functions using ratios of peak areas of caseinmacropeptide (CMP) and two other CMP-like degradation products were defined. The interference of proteolysis in the detection was estimated in samples adulterated on purpose with rennet whey, UHT treated in a pilot plant, and stored at 10, 20 and 30°C for up to 150 days. The application of the classification functions obtained allowed the detection of rennet whey solids added to milk. Only interferences due to very severe proteolysis, occurring after very long storage periods and/or at storage temperatures above room temperature, were observed.     

Proteolysis and storage stability of UHT milk produced in Turkey

The effect of raw milk quality (total and psychrotrophic bacterial and somatic cell counts, proteinase and plasmin activity) and UHT temperature (145 or 150 °C for 4 s) on proteolysis in UHT milk processed by a direct (steam-injection) system was investigated during storage at 25 °C for 180 d. High proteinase activity was measured in low-quality raw milk, which had high somatic cell count, bacterial count and plasmin activity. The levels of 12% trichloroacetic acid–soluble and pH 4.6-soluble nitrogen in all milk samples increased during storage, and samples produced from low-quality milk at the lower UHT temperature (145 °C) showed the highest values. Bitterness in UHT milk processed from low-quality milk at 145 °C increased during storage; gelation occurred in that milk after 150 d. The RP-HPLC profiles of pH 4.6-soluble fraction of the UHT milk samples produced at 150 °C showed quite small number of peaks after 180 d of storage. Sterilization at 150 °C extended the shelf-life of the UHT milk by reducing proteolysis, gelation and bitterness.     

Age gelation in UHT-processed reconstituted concentrated skim milk

UHT reconstituted concentrated skim milks made from high-heat powder had considerably longer gelation times than those made from medium- or low-heat powders. Addition of hexametaphosphate to the concentrated milk before UHT processing markedly delayed the onset of gelation during storage. Sediment formation was greatest in the UHT concentrated skim milk made using high-heat powder followed by samples made using medium- and low-heat powders, respectively. The extent of proteolysis, as measured by 12% TCA-soluble amino groups, increased at a faster rate in the UHT milks stored at 40°C than in those stored at 22°C but decreased with increasing heat treatment of the milk prior to powder manufacture. The electrophoretic patterns of samples stored at 22°C clearly showed the breakdown of β-casein with a corresponding increase in slower moving bands, presumably γ-casein and proteose-peptone components. However, storage of samples at 40°C resulted in diffused 'blurred' protein patterns with some protein material not entering the resolving gel. At 22°C there was some evidence of proteolysis but no evidence of high molecular weight polymer formation, while at 40°C both proteolysis and high molecular weight polymer formation increased with storage time. It appeared that both physico-chemical and proteolytic processes play some part in the mechanism of gelation in UHT reconstituted concentrated skim milk.     

UHT Milk Processing and Effect of Plasmin Activity on Shelf Life: A Review

Abstract: The demand for ultra‐high‐temperature (UHT) processed and aseptically packaged milk is increasing worldwide. A rise of 47% from 187 billion in 2008 to 265 billon in 2013 in pack numbers is expected. Selection of UHT and aseptic packaging systems reflect customer preferences and the processes are designed to ensure commercial sterility and acceptable sensory attributes throughout shelf life. Advantages of UHT processing include extended shelf life, lower energy costs, and the elimination of required refrigeration during storage and distribution. Desirable changes taking place during UHT processing of milk such as destruction of microorganisms and inactivation of enzymes occur, while undesirable effects such as browning, loss of nutrients, sedimentation, fat separation, cooked flavor also take place. Gelation of UHT milk during storage (age gelation) is a major factor limiting its shelf life. Significant factors that influence the onset of gelation include the nature of the heat treatment, proteolysis during storage, milk composition and quality, seasonal milk production factors, and storage temperature. This review is focused on the types of age gelation and the effect of plasmin activity on enzymatic gelation in UHT milk during a prolonged storage period. Measuring enzyme activity is a major concern to commercial producers, and many techniques, such as enzyme‐linked immunosorbent assay, spectrophotometery, high‐performance liquid chromatography, and so on, are available. Extension of shelf life of UHT milk can be achieved by deactivation of enzymes, by deploying low‐temperature inactivation at 55 °C for 60 min, innovative steam injection heating, membrane processing, and high‐pressure treatments.     

Composition and calcium status of acid whey from pasteurized,UHT‐treated and in‐bottle sterilized milks

Whey extracts were obtained from pasteurized, UHT‐treated and in‐bottle sterilized milks. After acidic precipitation of casein the concentration of protein, NPN, lactose, lipid, calcium, magnesium and potassium was determined. Among the parameters examined, protein content was significantly reduced in the whey extracts from UHT‐treated and in‐bottle sterilized milks compared with that from pasteurized milk, while lactose content was increased. Calcium extracted in whey was at least 80% of total calcium of the milk. The total calcium to protein ratio of whey was increased as a function of the thermal treatment of milk, while ionic calcium was about 50% of total calcium in all whey extracts. In vitro protein digestibility was found to be significantly lower in whey from UHT‐treated and in‐bottle sterilized milks than in that from pasteurized milk. Parallel estimation of the percentage of ionic calcium and of the solubility of proteins in the pH range 2–10 indicated that calcium was not involved in the pH‐dependent solubility of proteins extracted in the whey, the extent of solubility being essentially a function of the thermal treatment of milk. The results suggest that calcium was not responsible for the formation of soluble protein macroaggregates with impaired digestibility that are present in whey from milk subjected to heat treatment of increasing intensity.     

Development of an improved extraction and HPLC method for the measurement of ascorbic acid in cows' milk from processing plants and retail outlets

An improved extraction (2.5% HPO₃, 5 mm dithiothreitol) and HPLC quantification methodology using a C–18 column at 35 °C and 0.1 m acetic acid (98%) and acetonitrile (2%) mobile phase was developed to quantify total ascorbic acid (AA) in commercial whole/semi‐skim/skim raw/pasteurised/UHT milk packaged in opaque bags, transparent plastic, cardboard and Tetra Brik^?. AA content ranged from 0.21 to 10 and from 3.4 to 16 mg L^?1 in milk from retail outlets and processing plants, respectively, and was higher in organic milk. For same processor/lot samples, pasteurised milk showed higher AA content than UHT milk. This was not true for retail outlets samples. AA content was similar for whole/semi‐skim and semi‐skim/skim milk, but not for whole/skim comparisons. Among UHT samples, the AA content trend was whole<semi‐skim<skim and lower for UHT milk in opaque plastic and Tetra Brik^? container. After 14 days at 4 °C in the dark, AA losses ranged 35–83% depending on milk type and preservation method with a higher AA retention in unopened containers.     

A survey of the physicochemical and microbiological quality of ultra‐heat‐treated whole milk in Brazil during their shelf life

The objective was to evaluate the quality of important brands of ultra‐heat‐treated (UHT) milk marketed in the northern of Parana State, Brazil, during the shelf life of the product. Five brands were analysed at 30, 60, 90 and 120 days after UHT milk production. The physicochemical quality of the milk was assessed in terms of fat content, titratable acidity, density, freezing point depression, total solids, solids‐not‐fat, proteolysis and alcohol stability. Microbiological quality was assessed by the enumeration of aerobic mesophilic micro‐organisms. None of the brands complied with all the physicochemical standards. Two brands met all applicable microbiological requirements. Gelation was observed from 90 days of storage onwards in all brands.     

Determination of whey protein to total protein ratio in UHT milk using fourth derivative spectroscopy

A fourth derivative spectroscopy method was applied for the quantification of whey protein to total protein ratio in UHT milks. Some analytical features such as model compounds, selection of wavelength, linearity, repeatability and interference of milk fat were studied. The effect of refrigerated storage of raw milk, UHT treatment, and storage of UHT milk at room temperature on whey protein to total protein ratio was evaluated. No significant (p<0.05) differences among samples were found in any case. The ratio of whey protein to total protein was also determined in batches of whole (n=28) and skimmed (n=27) commercial UHT milks from different Spanish geographic areas processed by direct or indirect UHT systems in different periods of the year. The mean value was 18.1% for both whole and UHT skimmed milks. The analysis of laboratory-made mixtures of UHT milk with acid and rennet whey (2.5–15% of whey in milk expressed in protein) indicated that adulterations of UHT milk with whey up from 5% could be detected by the proposed method.     

Assessment of aflatoxin M1 levels in selected dairy products in north‐western Iran

The purpose of this survey was to evaluate the natural occurrence and content of aflatoxin M_1, AFM_1, in dairy products marketed in Urmia. During September 2007, 40 samples of pasteurised milk, 40 samples of ultra high temperature‐treated (UHT) milk, 40 samples of creamy cheese and 40 samples of Iranian Feta cheese were collected from different supermarkets in Urmia city. AFM₁ contents were determined by the competitive enzyme‐linked imunosorbent assay (ELISA) technique. All milk samples analysed showed a mean of AFM₁ concentrations lower than the permissible level of 50 ng/kg in Iran (23.22 and 19.53 ng/kg in pasteurised milk and UHT milk respectively). The mean levels of AFM₁ contamination were 43.31 ng/kg in Feta cheeses and 21.96 ng/kg in creamy cheeses. The potential risk of human exposure to aflatoxin M₁ via consumption of milk and milk products is well known. Dairy products must therefore be evaluated for aflatoxin and kept free from fungal contamination as much as possible.     

Determination of clenbuterol in UHT milk in Turkey

Clenbuterol use is linked to its ability to induce weight gain and a greater proportion of muscle to fat. Clenbuterol residues can affect lung and heart function in persons who have eaten liver or meat of animals which were given the drug. Any residue of clenbuterol is regarded unacceptable in the EU. The purpose of the study was to determine the occurrence of clenbuterol in UHT milk samples in Turkey. The occurrence of hormone residues in Ultra Heat Treatment (UHT) milk samples was investigated by semi‐quantitative enzyme‐linked immunoassay technique. There was a high incidence rate of clenbuterol, with forty‐one (68.3%) milk samples being contaminated. 21.7% of the samples were over the permissible level for clenbuterol as accepted by the EU. Clenbuterol levels in the samples purchased in Central Anatolia Region appear to be serious public health problem at the moment.     

Enhanced inactivation of bacterial lipases and proteinases in whole milk by a modified ultra high temperature treatment

Cultures of Pseudomonas spp. strains P10, P12 and P15 grown in whole milk which contained approximately 1 x 10(8) viable bacteria ml-1 demonstrated near linear increases in the concentration of short-chain free fatty acids and trichloroacetic acid soluble free amino groups at 20 degrees C, following either ultra high temperature (UHT) treatment (140 degrees C for 5 s) or dual heat treatments (140 degrees C followed by either 57, 60 or 65 degrees C). The dual heat treatments reduced the rates of lipolysis and proteolysis compared to the UHT treatment by up to 25-fold. The dual heat treatment utilizing 60 degrees C for 5 min also effectively limited both lipase and proteinase activities in raw milk culture samples which had contained either 6 x 10(6), 5 x 10(7) or 1 x 10(8) viable bacteria ml-1. In this system enzyme activities were reduced by up to 10-fold following dual heat treatment compared to UHT treatment alone.     

UHT纯牛奶消费喜好和关键感官特性的关联分析

以14种市场上不同类型超高温（Ultra-high temperature,UHT）纯牛奶为研究对象,采用9点喜好标度、适合项勾选法（Check-All-That-Apply,CATA）和恰好标度,同时结合偏最小二乘回归分析（PLS）分析不同类型UHT奶消费喜好和关键感官特性接受性差异。研究表明：不同类型UHT奶的整体喜好值范围为4.07~6.25,脱脂奶和部分全脂奶的喜好值最低（<5）,全脂和低脂奶的整体喜好差异不显著（p>0.05）。不同类型UHT奶的乳香、甜味、余味、浓稠和爽滑5个关键感官特性接受性上存在显著差异（p<0.05）,通过PLS回归模型筛选出导致消费喜好差异的7个关键感官特性接受性指标,分别为余味、乳香、甜味、浓稠、爽滑（-）、甜味（-）和乳香（-）。甜味和乳香偏弱以及爽滑度不够是分别导致低脂奶和部分全脂奶样品整体喜好偏低的关键感官特性。相关研究旨在为后期UHT纯牛奶消费喜好和产品感官品质研究提供基础理论和数据参考。     

Physicochemical,functional andATR‐FTIR molecular analysis of protein extracts derived from starchy pulses

An alkaline solubilisation and isoelectric point precipitation process were used to isolate proteins from broad bean, chickpea, lentil and white bean. The physicochemical, water solubility and foaming properties as well as their protein digestion characteristics of whole flours and its protein extracts were correlated with their molecular characteristics, analysed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). The processing of pulses resulted in protein recoveries ≥50.12% and yields up to 30.26% for broad bean. The purity of the protein was from 68.27% to 86.58% for chickpea and broad bean, with in vitro digestion values ≥80.59%. The protein extracts showed up to 0.85 mg g^?1 of phenolic compounds (gallic acid eq./100 g). The ATR‐FTIR analysis of the extracts showed different proportions of α‐helix and β‐sheet secondary structures of the amide II group. The analysis of the amide III group reflected inter‐ and intramolecular associations that could have influenced their emulsion and foam characteristics.     

On the influence of non‐enzymatic crosslinking of caseins on the gel strength of yoghurt

After storage of UHT milk at 37°C resp. 50°C, yoghurt was prepared. For a storage temperature of 37°C, breaking strength of the yoghurt samples increased from 2.7 to 5.8 N with increasing storage duration of the UHT milk. A plateau is reached after 17 days of storage. This increase in breaking strength correlates with a significant increase in non‐reducible casein oligomerization from 14% for fresh UHT milk to 25% measured using size exclusion chromatography under reducing and denaturing conditions and calculated as sum of predominantly formed dimers and trimers at the total casein fraction. At a storage temperature of 50°C, a less increase in breaking strength from 2.7 to 4.6 N with a plateau after 17 days was observed while casein oligomerization increased to 63%. After acid hydrolysis, only lysinoalanine and histidinoalanine were detected in the caseinate samples via amino acid analysis. The quantified concentration of lysinoalanine and histidinoalanine could not explain the observed casein oligomerization. Thus, unknown crosslinked amino acids must have been formed during storage, inducing significant changes in the functional properties of milk proteins.     

Influence of thermal processing conditions on flavor stability in fluid milk: benzaldehyde

Flavor loss in dairy products has been associated with enzymatic degradation by xanthine oxidase. This study was conducted to investigate the influence of milk thermal processing conditions (or xanthine oxidase inactivation) on benzaldehyde stability. Benzaldehyde was added to whole milk which had been thermally processed at 4 levels: (1) none or raw, (2) high temperature, short time (HTST) pasteurization, (3) HTST pasteurization, additionally heated to 100 degrees C (PAH), and (4) UHT sterilized. Additionally, PAH and UHT milk samples containing benzaldehyde (with and without ferrous sulfate) were spiked with xanthine oxidase. Azide was added as an antimicrobial agent (one additional pasteurized sample without) and the microbial load (total plate count) was determined on d 0, 2, and 6. The concentration of benzaldehyde and benzoic acid in all milk samples were determined at d 0, 1, 2, 4, and 6 (stored at 5 degrees C) by gas chromatography/mass spectrometry in selective ion monitory mode. Over the 6-d storage period, more than 80% of the benzaldehyde content was converted (oxidized) to benzoic acid in raw and pasteurized milk, whereas no change in the benzaldehyde concentration was found in PAH or UHT milk samples. Furthermore, the addition of xanthine oxidase or xanthine oxidase plus ferrous sulfate to PAH or UHT milk samples did not result in benzaldehyde degradation over the storage period.